Investigation of the multifunctional gene AOP3 expands the regulatory network fine-tuning glucosinolate production in Arabidopsis.

Jensen LM, Kliebenstein DJ, Burow M - Front Plant Sci (2015)

Bottom Line:
In this study, we use transgenic plants in combination with natural variation to investigate the regulatory role of the AOP3 gene found in GS-AOP locus previously suggested to contribute to the regulation of glucosinolate defense compounds.Phenotypic analysis and QTL mapping in F2 populations with different AOP3 transgenes support that the enzymatic function and the AOP3 RNA both play a significant role in controlling glucosinolate accumulation.Furthermore, we find different loci interacting with either the enzymatic activity or the RNA of AOP3 and thereby extend the regulatory network controlling glucosinolate accumulation.

ABSTRACTQuantitative trait loci (QTL) mapping studies enable identification of loci that are part of regulatory networks controlling various phenotypes. Detailed investigations of genes within these loci are required to ultimately understand the function of individual genes and how they interact with other players in the network. In this study, we use transgenic plants in combination with natural variation to investigate the regulatory role of the AOP3 gene found in GS-AOP locus previously suggested to contribute to the regulation of glucosinolate defense compounds. Phenotypic analysis and QTL mapping in F2 populations with different AOP3 transgenes support that the enzymatic function and the AOP3 RNA both play a significant role in controlling glucosinolate accumulation. Furthermore, we find different loci interacting with either the enzymatic activity or the RNA of AOP3 and thereby extend the regulatory network controlling glucosinolate accumulation.

Figure 5: GS-ELONG and AOP3 interaction in Col-0 × Gie-0 F2 populations for control of leaf glucosinolate levels. Average glucosinolate levels for the interaction of MAM1 or MAM2 with presence and absence of AOP3. For total aliphatic glucosinolate levels with the active AOP3 (A) and the AOP3 RNA (B), for SC glucosinolate levels with the active AOP3 (C) and the RNA (D), and for LC glucosinolate levels for the active AOP3 (E) and the untranslatable AOP3(F). Significance of the main effects and interaction are depicted by P < 0.05*, P < 0.01**, and P < 0.001*** and letters indicate significance of posttest with a levels of P < 0.05. Bean plots show strip charts of the individual plants levels, the density, and the average. For the population segregating with the active AOP3 n = 16, n = 44, n = 45, and n = 134 for the four groups. For the population segregating with the untranslatable RNA n = 14, n = 63, n = 52, and n = 152. FW, fresh weight.

Mentions:
For a more explicit test of the functions of the different AOP3 constructs and how the gene may be affected by the segregating background, we split the populations based on the lines' AOP3 transgene status and the accumulation of C3/(C3 + C4) as previously. We then tested if the genotypes at GS-ELONG (MAM1 or MAM2 inferred by the C3/(C3 + C4) threshold) and at AOP3 are linked with altered glucosinolate levels using the genotypes as factors in a linear model (Figure 5, Table S4). In agreement with previous observations, there was a significant interaction between the presence of the AOP3 FL construct and the GS-ELONG status for total aliphatic glucosinolate accumulation (Figure 5). Interestingly, the untranslatable AOP3 and the enzymatically active AOP3 had different effects on different glucosinolates suggesting that they influence different parts of the pathway. The functional AOP3 enzyme led to higher SC glucosinolate levels (Figure 5A), but this effect was not significant for the untranslatable AOP3 (Figure 5B). The higher accumulation of SC glucosinolates was found in plants with the interaction of the active AOP3 and MAM2 i.e., C3 (Figure 5C). Thus, the enzymatically active AOP3 can regulate SC accumulation, when present in a network containing MAM2 and other yet unknown components. A similar effect on SC levels was not observed of the AOP3 RNA (Figure 5D). In contrast, both the active AOP3 and the untranslatable AOP3 show significant effect on LC glucosinolate accumulation. No significant interaction between AOP3 and GS-ELONG was observed for this effect, thus, the effect does not dependent on whether plants produce predominantly C3 or C4 glucosinolates (Figures 5E,F). Together, this suggests that the effect of AOP3 on LC glucosinolates is not caused by the enzymatic activity and the associated flux, but instead by the AOP3 RNA in specific genetic backgrounds.

Figure 5: GS-ELONG and AOP3 interaction in Col-0 × Gie-0 F2 populations for control of leaf glucosinolate levels. Average glucosinolate levels for the interaction of MAM1 or MAM2 with presence and absence of AOP3. For total aliphatic glucosinolate levels with the active AOP3 (A) and the AOP3 RNA (B), for SC glucosinolate levels with the active AOP3 (C) and the RNA (D), and for LC glucosinolate levels for the active AOP3 (E) and the untranslatable AOP3(F). Significance of the main effects and interaction are depicted by P < 0.05*, P < 0.01**, and P < 0.001*** and letters indicate significance of posttest with a levels of P < 0.05. Bean plots show strip charts of the individual plants levels, the density, and the average. For the population segregating with the active AOP3 n = 16, n = 44, n = 45, and n = 134 for the four groups. For the population segregating with the untranslatable RNA n = 14, n = 63, n = 52, and n = 152. FW, fresh weight.

Mentions:
For a more explicit test of the functions of the different AOP3 constructs and how the gene may be affected by the segregating background, we split the populations based on the lines' AOP3 transgene status and the accumulation of C3/(C3 + C4) as previously. We then tested if the genotypes at GS-ELONG (MAM1 or MAM2 inferred by the C3/(C3 + C4) threshold) and at AOP3 are linked with altered glucosinolate levels using the genotypes as factors in a linear model (Figure 5, Table S4). In agreement with previous observations, there was a significant interaction between the presence of the AOP3 FL construct and the GS-ELONG status for total aliphatic glucosinolate accumulation (Figure 5). Interestingly, the untranslatable AOP3 and the enzymatically active AOP3 had different effects on different glucosinolates suggesting that they influence different parts of the pathway. The functional AOP3 enzyme led to higher SC glucosinolate levels (Figure 5A), but this effect was not significant for the untranslatable AOP3 (Figure 5B). The higher accumulation of SC glucosinolates was found in plants with the interaction of the active AOP3 and MAM2 i.e., C3 (Figure 5C). Thus, the enzymatically active AOP3 can regulate SC accumulation, when present in a network containing MAM2 and other yet unknown components. A similar effect on SC levels was not observed of the AOP3 RNA (Figure 5D). In contrast, both the active AOP3 and the untranslatable AOP3 show significant effect on LC glucosinolate accumulation. No significant interaction between AOP3 and GS-ELONG was observed for this effect, thus, the effect does not dependent on whether plants produce predominantly C3 or C4 glucosinolates (Figures 5E,F). Together, this suggests that the effect of AOP3 on LC glucosinolates is not caused by the enzymatic activity and the associated flux, but instead by the AOP3 RNA in specific genetic backgrounds.

Bottom Line:
In this study, we use transgenic plants in combination with natural variation to investigate the regulatory role of the AOP3 gene found in GS-AOP locus previously suggested to contribute to the regulation of glucosinolate defense compounds.Phenotypic analysis and QTL mapping in F2 populations with different AOP3 transgenes support that the enzymatic function and the AOP3 RNA both play a significant role in controlling glucosinolate accumulation.Furthermore, we find different loci interacting with either the enzymatic activity or the RNA of AOP3 and thereby extend the regulatory network controlling glucosinolate accumulation.

ABSTRACTQuantitative trait loci (QTL) mapping studies enable identification of loci that are part of regulatory networks controlling various phenotypes. Detailed investigations of genes within these loci are required to ultimately understand the function of individual genes and how they interact with other players in the network. In this study, we use transgenic plants in combination with natural variation to investigate the regulatory role of the AOP3 gene found in GS-AOP locus previously suggested to contribute to the regulation of glucosinolate defense compounds. Phenotypic analysis and QTL mapping in F2 populations with different AOP3 transgenes support that the enzymatic function and the AOP3 RNA both play a significant role in controlling glucosinolate accumulation. Furthermore, we find different loci interacting with either the enzymatic activity or the RNA of AOP3 and thereby extend the regulatory network controlling glucosinolate accumulation.